WO2019205026A1 - β-葡萄糖苷酶的用途及用其制备宝藿苷Ⅰ的方法 - Google Patents

β-葡萄糖苷酶的用途及用其制备宝藿苷Ⅰ的方法 Download PDF

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WO2019205026A1
WO2019205026A1 PCT/CN2018/084475 CN2018084475W WO2019205026A1 WO 2019205026 A1 WO2019205026 A1 WO 2019205026A1 CN 2018084475 W CN2018084475 W CN 2018084475W WO 2019205026 A1 WO2019205026 A1 WO 2019205026A1
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glucosidase
saponin
preparing
solution
epimedium
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PCT/CN2018/084475
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English (en)
French (fr)
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傅荣昭
刘立辉
曹磊
刘滔滔
郭杏林
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邦泰生物工程(深圳)有限公司
江西邦泰绿色生物合成生态产业园发展有限公司
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Priority to CN201880038008.3A priority Critical patent/CN110770351A/zh
Priority to PCT/CN2018/084475 priority patent/WO2019205026A1/zh
Publication of WO2019205026A1 publication Critical patent/WO2019205026A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin

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  • the invention relates to the field of bioengineering technology, in particular to a method for preparing a taxoside I by using a biological enzyme catalytic technique.
  • Baodiside I is a polyhydroxyflavonoid monomer compound in Epimedium, and is also an active ingredient in Epimedium.
  • Epimedium is a perennial herb of the genus Epimedium, a traditional Chinese medicine with a history of more than two thousand years. Modern pharmacological studies have shown that Epimedium has broad pharmacological effects such as enhancing immunity, improving cardiovascular system function, anti-inflammatory, anti-tumor, and promoting bone cell proliferation.
  • the most important medicinal ingredients in Epimedium are flavonoids, including icariin, pilosudin A, pilosudin B, radixidine C, arrowside A, arrowside B, and baicalin I. More than 200 ingredients, called Epimedium total flavonoids.
  • the extract of Epimedium sagittatum was extracted from Epimedium medicinal herbs by modern Chinese medicine extraction technology to obtain Epimedium extract, which gradually became an important raw material for many traditional Chinese medicine preparations.
  • Baojig I has unique pharmacological activity, has significant inhibitory effect on cancer cells, and can induce apoptosis of cancer cells. Its anticancer activity and biopharmaceutical properties are significantly better than Epimedium. Other flavonoids.
  • pharmacokinetic studies have found that icariin has been metabolized in the intestine to icariin and baumannin I before being absorbed by the body, and substances that absorb and enter the blood circulation to exert pharmacological effects are mainly capable of It is good to be absorbed by the body of protamine I. Therefore, the preparation and acquisition of the baicalin I monomer has gradually become the focus of researchers.
  • Breguet I can be directly extracted from Epimedium medicinal herbs.
  • the content of icariin is the highest in Epimedium, followed by the content of Asarum (A, B, C), and Baoji
  • the content of I is extremely low, only about 0.17 mg / g, and the structure and polarity of many components of epimedium I and epimedium are similar, which makes the carotenoid I monomer isolated from the drug of Epimedium It is extremely difficult, and the steps are cumbersome and the yield is low, which is not suitable for industrial applications.
  • the existing methods for preparing Baoji I are mostly prepared by hydrolyzing icariin with biological enzymes (such as ⁇ -glucosidase, cellulase, etc.), such as Chinese invention patent application CN 103160553 A, CN 102311985 A and CN.
  • 106148454 A is prepared by using high-purity icariin as a substrate.
  • the price of icariin monomer is higher, resulting in higher preparation costs.
  • a method of directly preparing the saponin I by using the extract of Epimedium (the total flavonoids of Epimedium) as a substrate and catalyzing the preparation of the saponin I.
  • this preparation method usually only converts icariin in Epimedium extract into Baoji I, and cannot simultaneously convert some other components in Epimedium extract into Baoji I.
  • the extract of Epimedium does not achieve maximum effective use.
  • the object of the present invention is to solve the technical problem of failing to maximize the effective utilization of Epimedium extract by the existing method for preparing the Biosynthesis of Epimedium extract mentioned in the above background art.
  • Providing a novel method for enzymatically preparing Baoji I which can not only convert icariin in epimedium extract into protamine I, but also simultaneously extract other epimedium extracts Some of the ingredients are also converted into Baoji I, which improves the utilization of Epimedium extract and reduces the preparation cost of Baoling I.
  • a method for preparing a novel baicalin I is provided, in particular, the hydrolysis of a substrate is catalyzed by ⁇ -glucosidase to prepare a saponin I, the substrate is epimedium extract.
  • Epimedium extract refers to the ethanol extract of the dried stems and leaves of Epimedium, which is mainly composed of flavonoids in Epimedium, also known as Epimedium flavonoids.
  • the method for preparing the saponin I provided by the present invention is suitable for the extract of icariin having an icariin content ranging from 10% to 50%.
  • the ⁇ -glucosidase has a nucleotide sequence as shown in SEQ ID NO: 1.
  • the reaction system for preparing the taxoside I by catalyzing the hydrolysis of the substrate by ⁇ -glucosidase further comprises a buffer solution and a co-solvent, wherein the buffer has a pH of 5.0-7.0, and the reaction system is controlled during the reaction.
  • the temperature is from 45 ° C to 80 ° C
  • the pH is from 4.5 to 6.5
  • the reaction time is from 2 to 10 h.
  • the temperature of the reaction system is controlled during the reaction from 45 ° C to 60 ° C, the pH is from 4.5 to 6.0, and the reaction time is from 6 to 10 h.
  • the buffer is used in an amount of 60% to 90% (v/v) of the total reaction liquid, and the auxiliary solvent is used in an amount of 5% to 20% (v/v) of the total reaction liquid.
  • the buffer is an acetate buffer.
  • the co-solvent is selected from any one of acetone, methanol, ethanol, NMP, Tween 20, and Tween 80.
  • the co-solvent is acetone
  • acetone is selected for post-treatment operation (ie, the enzyme-catalyzed reaction solution after the end of the enzyme-catalyzed reaction)
  • the advantage of separating the operation of the saponin I) is simple.
  • the ⁇ -glucosidase used in the above methods include liquid enzyme solutions, solids, and various immobilized enzymes, either in the form of unpurified crude enzymes or in partially or completely purified form.
  • the ⁇ -glucosidase is added in the form of a crude enzyme solution, which is obtained by inducing expression by a microorganism strain containing the ⁇ -glucosidase gene, and then removing the precipitate by centrifugation and centrifugation.
  • a solution containing ⁇ -glucosidase obtained.
  • the crude enzyme solution is used in an amount of 5% to 20% (v/v) of the total reaction liquid, and the substrate concentration is from 1% to 10% (w/v) of the total reaction liquid.
  • the preparation process of the crude enzyme solution comprises: constructing a recombinant plasmid containing the ⁇ -glucosidase gene, transferring the recombinant plasmid into a microorganism strain, culturing the microorganism strain, and performing induced expression.
  • ⁇ -glucosidase after which the microbial strain is collected in a buffer, and the crude enzyme solution is obtained by cell disruption and centrifugation to remove the precipitate.
  • the microbial strain is Escherichia coli Rosetta (DE3).
  • the beryoside I monomer needs to be separated from the enzyme-catalyzed reaction solution, and the obtained enzyme-catalyzed reaction solution is preferably crystallized.
  • the process is to collect the crude crystal, which is the crude product of the saponin I.
  • the crystallization process comprises: distilling off the acetone in a 45-55 ° C water bath environment under reduced pressure, and after 9/10 of the acetone is distilled off, Then, the remaining solution is cooled to 10-15 ° C, and after all the crystals are precipitated, the mixture is filtered, and the filter cake is washed with water and dried to obtain a crude product of protamine I.
  • the above-described cooling process is maintained in a water bath environment.
  • the drying treatment means that the filter cake is dried in a 70 ° C oven to a moisture content of less than 5% in the crude Baoji I, and the drying time is preferably 12 h.
  • the recrystallization process comprises: dissolving the crude Baojiu I in anhydrous ethanol, adding activated carbon, stirring and heating to 55-65 ° C, filtering while hot, and rinsing with absolute ethanol.
  • the cake and the filtrate are distilled under reduced pressure to remove ethanol, and then water is added.
  • the whole process is maintained at a temperature of 55-65 ° C, then the temperature is lowered to 5-10 ° C, and stirred until the crystals are completely precipitated. After filtration, the filter cake is washed with an ethanol solution and dried to obtain a protamine I product.
  • the mixture is stirred at 55-65 ° C for at least 1 h before the hot filtration.
  • the process of adding water should be kept slowly dropping.
  • the crystallization process should be slowly stirred.
  • the ethanol solution is selected from a cold ethanol aqueous solution having a volume ratio of 40%.
  • the ethanol solution used for the second time is preferably a cold ethanol aqueous solution having a volume ratio of 60%.
  • the present invention provides a ⁇ -glucosidase and a microbial strain containing the ⁇ -glucosidase gene in catalyzing epimedium extract, icariin, pilosudation A, pilgrin B,
  • a ⁇ -glucosidase and a microbial strain containing the ⁇ -glucosidase gene in catalyzing epimedium extract, icariin, pilosudation A, pilgrin B
  • the ⁇ -glucosidase has the amino acid sequence shown in SEQ ID NO:
  • the ⁇ -glucosidase specifically hydrolyzes the icariin, the diarrhea A, the carbamazepine B, the arrowside A, and the arrowhead B in the extract of Epimedium into Baodi Glycoside I.
  • the ⁇ -glucosidase has a nucleotide sequence as shown in SEQ ID NO: 1.
  • the preparation method of the saponin I provided by the invention has the following advantages:
  • the ⁇ -glucosidase known in the prior art generally only has the function of hydrolyzing glucosidic bonds, so usually only the icariin can be hydrolyzed to the saponin I.
  • the present invention screens a ⁇ -glucosidase having various functions, and when the extract of the extract of Epimedium is used as a substrate, the icariin can be hydrolyzed to the valerian I, and At the same time, it can simultaneously hydrolyze the radix A, the sputum B, the scorpion A and the arrow saponin B to the saponin I, thereby improving the effective utilization rate of the extract of Epimedium and greatly reducing the saponin I Preparation costs.
  • the invention adopts the crude enzyme solution as the source of ⁇ -glucosidase, and can rapidly prepare the crude enzyme solution by expressing in E. coli, which greatly reduces the preparation cost.
  • the macroporous resin is generally used to separate and purify the saponin I monomer, and the process is complicated and the cost is high.
  • the method provided by the invention uses the crystallization and recrystallization process to separate and purify the Baojiu I monomer, thereby reducing the cost, and the process is simple and easy to operate, and the efficiency is high.
  • Figure 1 is an HPLC chromatogram of icariin standard
  • Figure 2 is an HPLC chromatogram of the standards of ⁇ A, ⁇ B, ⁇ C, and Baojiin I;
  • Figure 3 is an HPLC chromatogram of the substrate extract of Epimedium used in Example 2 of the present invention.
  • Figure 4 is an HPLC chart of the enzyme-catalyzed reaction solution of Example 2 of the present invention.
  • Figure 5 is an HPLC chart of an enzyme-catalyzed reaction solution of sputum A in Example 5 of the present invention
  • Figure 6 is an HPLC chart of an enzyme-catalyzed reaction solution of phlegm-B in Example 5 of the present invention.
  • Fig. 7 is an HPLC chart of an enzyme-catalyzed reaction solution of sputum C in Example 5 of the present invention.
  • the ⁇ -glucosidase gene (GenBank Accession No. AGS52251.1 , the present invention is numbered BGL14, the nucleotide sequence of which is shown in SEQ ID NO: 1, and the amino acid sequence thereof is shown in SEQ ID NO: 2) is linked to The recombinant plasmid pET22b-BGL14 was obtained between the Nde I site and the EcoR I site of the expression vector pET22b(+). The recombinant plasmid was transferred into E.
  • coli Rosetta (DE3) competent cells and the recombinant plasmid-containing Escherichia coli was inoculated into 5 mL of LB medium (containing 100 ⁇ g/mL of Amp), and cultured at 37 ° C, shaking at 200 rpm overnight. Transfer 1% of the inoculum to 100-1000 mL of LB medium (containing 100 ⁇ g/mL of Amp), incubate at 37 ° C, shaker at 200 rpm until the OD600 reaches 0.6-1.0, and add a final concentration of 0.1-0.5 mM.
  • IPTG Propyl- ⁇ -D-thiogalactoside
  • Epimedium extract produced by Shaanxi Huike Plant Development Co., Ltd. was used, which contained 20% by weight of icariin and a total of 12% of Chaoyang Ding, Chaoyang Ding B. And ⁇ C, its HPLC map is shown in Figure 3.
  • Epimedium extract 100 g was weighed into a 2 L three-necked flask, 780 ml of acetate buffer (pH 6.0) was added, 100 ml of acetone was added, and after stirring, the ⁇ -glucosidase prepared in Example 1 was added.
  • the enzyme solution was 120 ml, the pH was controlled at 6.0, and the reaction was started at a temperature of 45 °C. After 6 h of reaction, the enzyme-catalyzed reaction solution was subjected to HPLC detection.
  • the detection conditions were: Xunxu Xtimate C18 5 ⁇ m ⁇ 250 ⁇ 4.6 mm column, wavelength UV 270 nm, mobile phase 90% acetonitrile aqueous solution, flow rate 1.0 ml/min, temperature 25 ° C, The resulting HPLC profile is shown in Figure 4.
  • the enzyme-catalyzed reaction solution is placed in a 50 ° C water bath for distillation under reduced pressure, and most of the acetone (about 90 ml) is distilled off, and the remaining solution is placed in a water bath and stirred to cool to 10-15 ° C. . Filtration, the aqueous solution was filtered off, the filter cake was rinsed with 50 ml of water, and the filter cake was dried in an oven at 70 ° C for 12 h to ensure that the moisture content was less than 5%, and 104.5 g of crude Baojig I was obtained, the purity was 70.3%, and the content was 15.35. %.
  • the crude Baojiu I prepared in Example 3 was dissolved in 250 ml of absolute ethanol, 2 g of activated carbon was added, stirred and heated to 60 ° C for 1 h, filtered while hot, the filter cake was rinsed with 20 ml of ethanol, and the filtrate was placed under a water bath at 60 ° C.
  • the detection conditions were as follows: Yuexu Xtimate C18 5 ⁇ m ⁇ 250 ⁇ 4.6 mm column, wavelength UV 270 nm, mobile phase 90% acetonitrile aqueous solution, flow rate 1.0 ml/min, temperature 25 °C.
  • the HPLC chromatograms of the enzyme-catalyzed reaction solutions of Asarum A, Asarum B and Asarum C are shown in Figures 5, 6 and 7, respectively.
  • the test results showed that both C. sinensis and C. sylvestris B were 100% converted into scutellarin I, and all of radix was determined to be rhamnosyl icariin II.
  • the sources of the ⁇ -glucosidase genes of the crude enzyme solutions numbered 1-10 are Aspergillus oryzae RIB40, Aspergillus fumigatus A1163, Aspergillus niger, Talaromyces leycettanus JCM12802, Thermotoga maritima MSB8, Aspergillus terreus, Aspergillus ustus, Aspergillus oryzae, Dictyoglomus thermophilum DSM 3960, Caldanaerobius fijiensis.

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Abstract

提供了一种使用β-葡萄糖苷酶制备宝藿苷Ⅰ的方法，包括用β-葡萄糖苷酶催化底物水解制备宝藿苷Ⅰ，底物为淫羊藿提取物、淫羊藿苷、朝藿定A、朝藿定B、箭藿苷A和箭藿苷B中的任意一种或多种，所述β-葡萄糖苷酶具有如SEQ ID NO：2所示的氨基酸序列。该方法能够将淫羊藿、朝藿定A、朝藿定B、箭藿苷A及箭藿苷B全部水解成宝藿苷Ⅰ，提高了淫羊藿提取物的有效利用率，大大降低了宝藿苷Ⅰ的制备成本。还提供了所述β-葡萄糖苷酶以及含有所述β-葡萄糖苷酶基因的微生物菌株在催化淫羊藿提取物、淫羊藿苷、朝藿定A、朝藿定B、箭藿苷A和箭藿苷B中的任意一种或多种水解，制备宝藿苷Ⅰ中的应用。

Description

β-葡萄糖苷酶的用途及用其制备宝藿苷Ⅰ的方法 技术领域
本发明涉及生物工程技术领域，特别涉及一种利用生物酶催化技术制备宝藿苷Ⅰ的方法。
背景技术
宝藿苷Ⅰ是淫羊藿中的一种多羟基黄酮类单体化合物，也是淫羊藿药材中的一种有效成分。淫羊藿为小檗科淫羊藿属多年生草本植物，是具有两千多年用药历史的传统中药。现代药理研究表明，淫羊藿具有增强免疫力、改善心血管系统功能、抗炎、抗肿瘤、促进骨细胞增殖等药理作用，具有广阔的用药前景。淫羊藿中最为重要的药效成分是黄酮类化合物，包括淫羊藿苷、朝藿定A、朝藿定B、朝藿定C、箭藿苷A、箭藿苷B、宝藿苷Ⅰ等200多种成分，称为淫羊藿总黄酮。采用现代中药提取技术将淫羊藿总黄酮从淫羊藿药材中提取出来，得到淫羊藿提取物，逐渐成为众多中药制剂的重要原料。
近年来，国内外研究发现，宝藿苷Ⅰ的药理活性独特，对癌细胞具有显著的抑制作用，并可诱导癌细胞凋亡，其抗癌活性和生物药剂学性质明显优于淫羊藿中其他黄酮类化合物。同时，药代动力学研究发现，淫羊藿苷在被人体吸收之前，已经在肠道中代谢成了淫羊藿素和宝藿苷I，吸收并进入血液循环发挥药理效应的物质主要是能更好被机体吸收的宝藿苷Ⅰ。因此，宝藿苷Ⅰ单体的制备和获取逐渐成为广大研究者关注的重点。
宝藿苷Ⅰ可以从淫羊藿药材中直接提取，然而研究发现，淫羊藿中以淫羊藿苷的含量最多，朝藿定(A、B、C)的含量次之，而宝藿苷Ⅰ的含量极低，只有约0.17mg/g，并且宝藿苷Ⅰ与淫羊藿中的很多成分的结构和极性相近，这使得通过从淫羊藿药材中分离得到宝藿苷Ⅰ单体的难度极大，并且步骤繁琐，产率较低，不适于工业化应用。
现有的制备宝藿苷Ⅰ的途径多采用生物酶(如β-葡萄糖苷酶、纤维素酶等)水解淫羊藿苷的方法制备，如中国发明专利申请CN 103160553 A、 CN 102311985 A和CN 106148454 A都是以高纯度淫羊藿苷为底物制备宝藿苷Ⅰ。可是淫羊藿苷单体的价格较高，从而导致制备成本较高。为克服淫羊藿苷价格较高的问题，又有采用直接以淫羊藿提取物(淫羊藿总黄酮)为底物，用生物酶催化制备宝藿苷Ⅰ的方法。但是此种制备方法通常也只是将淫羊藿提取物中的淫羊藿苷转化成宝藿苷Ⅰ，而不能同时将淫羊藿提取物中的其他某些成分也转化成宝藿苷Ⅰ，从而使得淫羊藿提取物没有达到最大限度的有效利用。
发明内容
本发明的目的在于解决上述背景技术中提到的现有生物酶催化淫羊藿提取物制备宝藿苷Ⅰ的方法所存在的未能对淫羊藿提取物达到最大限度的有效利用的技术问题，提供一种酶法制备宝藿苷Ⅰ的新方法，该方法不仅能够将淫羊藿提取物中的淫羊藿苷转化成宝藿苷Ⅰ，而且能同时将淫羊藿提取物中的其他某些成分也转化成宝藿苷Ⅰ，提高了淫羊藿提取物的利用率，降低了宝藿苷Ⅰ的制备成本。
为实现上述目的，发明人经过长期大量的实验摸索，在筛选了几十种不同来源的β-葡萄糖苷酶后，终于筛选出一种可同时将淫羊藿提取物中的多种成分转化成宝藿苷Ⅰ的β-葡萄糖苷酶。基于此，本发明一方面提供了一种新的宝藿苷Ⅰ的制备方法，具体为，用β-葡萄糖苷酶催化底物水解制备宝藿苷Ⅰ，所述底物为淫羊藿提取物、淫羊藿苷、朝藿定A、朝藿定B、箭藿苷A和箭藿苷B中的任意一种或多种，所述β-葡萄糖苷酶具有如SEQ ID NO：2所示的氨基酸序列，且所述β-葡萄糖苷酶特异性地将所述淫羊藿提取物中的淫羊藿苷、朝藿定A、朝藿定B、箭藿苷A和箭藿苷B全部水解成宝藿苷Ⅰ。
淫羊藿提取物是指淫羊藿的干燥茎叶经加工制成的乙醇提取物，以淫羊藿中的黄酮类化合物为主要成分为，又名淫羊藿总黄酮。本发明提供的制备宝藿苷Ⅰ的方法适用于淫羊藿苷的含量范围为10％-50％的淫羊藿提取物。
优选地，所述β-葡萄糖苷酶具有如SEQ ID NO：1所示的核苷酸序列。
具体地，所述用β-葡萄糖苷酶催化底物水解制备宝藿苷Ⅰ的反应体系中还含有缓冲液和助溶剂，所述缓冲液的pH值为5.0-7.0，反应过程中控制反应体系的温度为45℃-80℃，pH值为4.5-6.5，反应时间为2-10h。
优选地，反应过程中控制反应体系的温度为45℃-60℃，pH值为4.5-6.0，反应时间为6-10h。
优选地，所述缓冲液的用量为占总反应液的60％-90％(v/v)，所述助溶剂的用量为占总反应液的5％-20％(v/v)。
优选地，所述缓冲液为醋酸盐缓冲液。
优选地，所述助溶剂选自丙酮、甲醇、乙醇、NMP、吐温20和吐温80中的任意一种。
更优选地，所述助溶剂为丙酮，相比较选用甲醇、乙醇、NMP、吐温20或吐温80作为助溶剂时，选用丙酮具有后处理操作(即酶催化反应结束后从酶催化反应液中分离宝藿苷Ⅰ的操作)简单的优点。
上述方法中所使用的β-葡萄糖苷酶的具体存在形式包括液态酶液、固态以及各种固定化酶，可以是未经纯化的粗酶形式，也可以是经部分纯化或完全纯化的形式。优选地，所述β-葡萄糖苷酶以粗酶液的形式投加，所述粗酶液是指由含有所述β-葡萄糖苷酶基因的微生物菌株诱导表达后经破胞、离心去除沉淀后获得的含有β-葡萄糖苷酶的溶液。优选地，所述粗酶液的用量为占总反应液的5％-20％(v/v)，所述底物浓度为占总反应液的1％-10％(w/v)。
具体地，所述粗酶液的制备过程包括：构建含有所述β-葡萄糖苷酶基因的重组质粒，并将所述重组质粒转入微生物菌株体内，培养所述微生物菌株并进行诱导表达所述β-葡萄糖苷酶，之后收集所述微生物菌株于缓冲液中，经破胞、离心去除沉淀后即得所述粗酶液。
优选地，所述微生物菌株为大肠杆菌Rosetta(DE3)。
待所述用β-葡萄糖苷酶催化底物水解制备宝藿苷Ⅰ的反应结束后，需要将宝藿苷Ⅰ单体从酶催化反应液中分离出来，优选将获得的酶催化反应液进行结晶工艺处理，收集结晶体即得宝藿苷Ⅰ粗品，为提高宝藿苷Ⅰ的 纯度，优选将所述宝藿苷Ⅰ粗品进行重结晶工艺处理，收集结晶体即得宝藿苷Ⅰ精制品。
具体地，当助溶剂为丙酮时，所述结晶工艺处理过程包括：将所述酶催化反应液于45-55℃水浴环境中减压蒸馏出丙酮，至9/10的丙酮被蒸馏出后，再将剩余溶液降温至10-15℃，待结晶体全部析出后过滤，滤饼经水洗、干燥处理后即得宝藿苷Ⅰ粗品。
优选地，上述降温过程保持在水浴环境中进行。
优选地，所述干燥处理是指将滤饼置于70℃烘箱中干燥至宝藿苷Ⅰ粗品中的水分含量低于5％，干燥时间优选为12h。
具体地，所述重结晶工艺处理过程包括：将所述宝藿苷Ⅰ粗品溶解于无水乙醇中，并加入活性炭，搅拌并加热至55-65℃，趁热过滤，并用无水乙醇冲洗滤饼，滤液经减压蒸馏出乙醇后再加水，优选待蒸馏出50％-70％的乙醇后加水，整个过程维持温度在55-65℃，之后降温至5-10℃，搅拌至结晶体全部析出后，过滤，滤饼经乙醇溶液冲洗、干燥处理后即得宝藿苷Ⅰ精制品。
优选地，为使活性炭对不溶性杂质的吸附完全，在趁热过滤之前，保持55-65℃下搅拌至少1h。
优选地，加水的过程应保持缓慢滴加。
优选地，析晶过程应缓慢搅拌。
优选地，所述乙醇溶液选用体积比为40％的冷乙醇水溶液。
为进一步提高宝藿苷Ⅰ精制品的纯度，优选将干燥处理之前的滤饼重复进行重结晶工艺处理，即可获得纯度进一步提高的宝藿苷Ⅰ精制品。
进一步地，重复进行重结晶工艺处理过程中，第二次使用的乙醇溶液优选为体积比为60％的冷乙醇水溶液。
另一方面，本发明还提供了β-葡萄糖苷酶以及含有所述β-葡萄糖苷酶基因的微生物菌株在催化淫羊藿提取物、淫羊藿苷、朝藿定A、朝藿定B、箭藿苷A和箭藿苷B中的任意一种或多种制备宝藿苷Ⅰ中的应用，其特征在于：所述β-葡萄糖苷酶具有如SEQ ID NO：2所示的氨基酸序列，且所 述β-葡萄糖苷酶特异性地将所述淫羊藿提取物中的淫羊藿苷、朝藿定A、朝藿定B、箭藿苷A和箭藿苷B全部水解成宝藿苷Ⅰ。
优选地，所述β-葡萄糖苷酶具有如SEQ ID NO：1所示的核苷酸序列。
有益效果：
与现有技术相比，本发明提供的宝藿苷Ⅰ的制备方法具有如下优点：
1、现有技术中已知的β-葡萄糖苷酶一般只具备水解葡萄糖苷键的作用，所以通常只能将淫羊藿苷水解成宝藿苷Ⅰ。本发明筛选到了一种具有多种功能的β-葡萄糖苷酶，在以淫羊藿提取物为底物制备宝藿苷Ⅰ时，不仅能够将淫羊藿苷全部水解成宝藿苷Ⅰ，而且能同时将朝藿定A、朝藿定B、箭藿苷A和箭藿苷B全部水解成宝藿苷Ⅰ，从而提高了淫羊藿提取物的有效利用率，大大降低了宝藿苷Ⅰ的制备成本。
2、本发明采用粗酶液作为β-葡萄糖苷酶的来源，通过在大肠杆菌中表达可以快速制备粗酶液，极大地降低了制备成本。
3、现有技术中普遍采用大孔树脂对宝藿苷Ⅰ单体进行分离和纯化，工艺复杂，成本较高。本发明提供的方法采用结晶和重结晶工艺对宝藿苷Ⅰ单体进行分离和纯化，降低了成本，且工艺简单易操作，效率高。
附图说明
图1是淫羊藿苷标准品的HPLC图谱；
图2是朝藿定A、朝藿定B、朝藿定C以及宝藿苷Ⅰ标准品的HPLC图谱；
图3是本发明实施例2中使用的底物淫羊藿提取物的HPLC图谱；
图4是本发明实施例2的酶催化反应液的HPLC图谱；
图5是本发明实施例5中朝藿定A的酶催化反应液的HPLC图谱；
图6是本发明实施例5中朝藿定B的酶催化反应液的HPLC图谱；
图7是本发明实施例5中朝藿定C的酶催化反应液的HPLC图谱。
具体实施方式
下面结合具体实施例和附图对本发明做进一步的详细说明，以下实施例是对本发明的解释，本发明并不局限于以下实施例，实施例中未注明具 体条件者，按常规条件或制造商建议的条件进行。若无特别说明，本发明实施例中所使用的原料及其它化学试剂皆为市售商品。
实施例1
β-葡萄糖苷酶粗酶液的制备。
将β-葡萄糖苷酶基因(GenBank登录号为 AGS52251.1，本发明编号为BGL14，其核苷酸序列如SEQ ID NO：1所示，其氨基酸序列如SEQ ID NO：2所示)连接到表达载体pET22b(+)的Nde I位点和EcoR I位点之间，得到重组质粒pET22b-BGL14。将重组质粒转入大肠杆菌Rosetta(DE3)感受态细胞中，将含有重组质粒的大肠杆菌接种在5mL的LB培养基(含有100μg/mL的Amp)，37℃、200rpm摇床培养过夜后，以1％的接种量转接到100-1000mL的LB培养基中(含有100μg/mL的Amp)，在37℃、200rpm摇床培养至OD600达到0.6～1.0，加入终浓度为0.1-0.5mM的异丙基-β-D-硫代半乳糖苷(IPTG)，在37℃、200rpm摇床培养8-10h后离心收集菌体。菌体用4倍体积的浓度为100mM的磷酸盐缓冲液(pH6.5)悬浮，用超声波或均质机破胞，离心去沉淀得到β-葡萄糖苷酶的粗酶液。
实施例2
宝藿苷Ⅰ的制备
以淫羊藿提取物为底物，选用陕西慧科植物开发有限公司生产的淫羊藿提取物，其含有20％重量的淫羊藿苷和总计12％的朝藿定A、朝藿定B和朝藿定C，其HPLC图谱如图3所示。
称取100g淫羊藿提取物置于2L的三口瓶中，加入780ml的醋酸盐缓冲液(pH6.0)，加入100ml的丙酮，搅拌均匀后，加入实施例1制备的β-葡萄糖苷酶粗酶液120ml，控制pH 6.0，温度45℃开始反应。反应6h后，取酶催化反应液进行HPLC检测，检测条件：月旭Xtimate C18 5μm×250×4.6mm色谱柱，波长UV270nm，流动相为90％乙腈水溶液，流速1.0ml/min，温度25℃，所得HPLC图谱如图4所示。
实施例3
宝藿苷Ⅰ粗品的制备
待实施例2的酶催化反应结束后，将酶催化反应液置于50℃水浴中减压蒸馏，蒸出大部分丙酮(约90ml)，剩余溶液置于水浴锅中搅拌降温至10-15℃。过滤，将水溶液滤出，用50ml水冲洗滤饼，滤饼置于70℃烘箱中干燥12h，确保水分含量低于5％，得到宝藿苷Ⅰ粗品104.5g，纯度为70.3％，含量为15.35％。
实施例4
宝藿苷Ⅰ精制品的制备
将实施例3制备的宝藿苷Ⅰ粗品用250ml无水乙醇溶解，加入2g活性炭，搅拌加热至60℃保持1h，趁热过滤，用20ml乙醇冲洗滤饼，滤液置于60℃水浴下减压蒸馏，蒸出170ml乙醇，剩余溶液转移至60℃水浴锅中，缓慢滴加150ml纯水，温度降至5-10℃，缓慢搅拌保持2h，过滤，用20ml40％冷乙醇冲洗滤饼，滤饼再次用200ml无水乙醇溶解，加入2g活性炭，60℃搅拌保持1h，过滤，用20ml乙醇冲洗滤饼，滤液减压浓缩，蒸出120ml乙醇，将剩余溶液置于60℃水浴锅，缓慢滴加54ml纯水，降温至5-10℃，缓慢搅拌保持2h，过滤，用20ml 60％乙醇冲洗滤饼，滤饼置于70℃烘箱干燥12h，确保水分含量低于1％，得到宝藿苷Ⅰ精制品13.26g。按水解后分子量减少的理论收率折算为86.18％。产品取样检测纯度为98.70％，含量为98.77％。
实施例5
称取朝藿定A、朝藿定B和朝藿定C各1mg分别置于2mL的离心管中，加入780μL的醋酸盐缓冲液(pH6.0)，加入100μL的丙酮，加入实施例1制备的β-葡萄糖苷酶粗酶液120μL，控制pH 6.0，温度45℃开始反应。反应6h后，取酶催化反应液进行HPLC检测，检测条件：月旭Xtimate C18 5μm×250×4.6mm色谱柱，波长UV270nm，流动相为90％乙腈水溶液，流速1.0ml/min，温度25℃。朝藿定A、朝藿定B和朝藿定C的酶催化反应液的HPLC图谱分别如图5、图6和图7所示。检测结果显示，朝藿定A和朝藿定B均100％转化成了宝藿苷Ⅰ，而朝藿定C全部转化成了鼠李糖基淫羊藿次苷Ⅱ。
对比例
参照实施例1分别制备另外10种不同来源的β-葡萄糖苷酶的粗酶液，并参照实施例2制备宝藿苷Ⅰ，检测每种粗酶液对底物的转化率如表1所示，表中，编号1-10的粗酶液的β-葡萄糖苷酶基因来源分别为Aspergillus oryzae RIB40、Aspergillus fumigatus A1163、Aspergillus niger、Talaromyces leycettanus JCM12802、Thermotoga maritima MSB8、Aspergillus terreus、Aspergillus ustus、Aspergillus oryzae、Dictyoglomus thermophilum DSM 3960、Caldanaerobius fijiensis。
表1
Figure PCTCN2018084475-appb-000001

Claims (12)

  1. 宝藿苷Ⅰ的制备方法，其特征在于：用β-葡萄糖苷酶催化底物水解制备宝藿苷Ⅰ，所述底物为淫羊藿提取物、淫羊藿苷、朝藿定A、朝藿定B、箭藿苷A和箭藿苷B中的任意一种或多种，所述β-葡萄糖苷酶具有如SEQ ID NO：2所示的氨基酸序列，且所述β-葡萄糖苷酶特异性地将所述淫羊藿提取物中的淫羊藿苷、朝藿定A、朝藿定B、箭藿苷A和箭藿苷B全部水解成宝藿苷Ⅰ。
  2. 根据权利要求1所述的宝藿苷Ⅰ的制备方法，其特征在于：所述β-葡萄糖苷酶具有如SEQ ID NO：1所示的核苷酸序列。
  3. 根据权利要求1或2所述的宝藿苷Ⅰ的制备方法，其特征在于：所述用β-葡萄糖苷酶催化底物水解制备宝藿苷Ⅰ的反应体系中还含有缓冲液和助溶剂，所述缓冲液的pH值为5.0-7.0，反应过程中控制反应体系的温度为45℃-80℃，pH值为4.5-6.5，反应时间为2-10h。
  4. 根据权利要求3所述的宝藿苷Ⅰ的制备方法，其特征在于：所述缓冲液的用量为占总反应液的60％-90％(v/v)，所述助溶剂的用量为占总反应液的5％-20％(v/v)。
  5. 根据权利要求3所述的宝藿苷Ⅰ的制备方法，其特征在于：所述助溶剂选自丙酮、甲醇、乙醇、NMP、吐温20和吐温80中的任意一种。
  6. 根据权利要求1或2所述的宝藿苷Ⅰ的制备方法，其特征在于：所述β-葡萄糖苷酶以粗酶液的形式投加，所述粗酶液是指由含有所述β-葡萄糖苷酶基因的微生物菌株诱导表达后经破胞、离心去除沉淀后获得的含有β-葡萄糖苷酶的溶液。
  7. 根据权利要求6所述的宝藿苷Ⅰ的制备方法，其特征在于，所述粗酶液的制备过程包括：构建含有所述β-葡萄糖苷酶基因的重组质粒，并将所述重组质粒转入微生物菌株体内，培养所述微生物菌株并进行诱导表达所述β-葡萄糖苷酶，之后收集所述微生物菌株于缓冲液中，经破胞、离心去除沉淀后即得所述粗酶液。
  8. 根据权利要求6所述的宝藿苷Ⅰ的制备方法，其特征在于：所述粗酶液的用量为占总反应液的5％-20％(v/v)，所述底物浓度为占总反应液的 1％-10％(w/v)。
  9. 根据权利要求3所述的宝藿苷Ⅰ的制备方法，其特征在于：待所述用β-葡萄糖苷酶催化底物水解制备宝藿苷Ⅰ的反应结束后，将获得的酶催化反应液进行结晶工艺处理，收集结晶体即得宝藿苷Ⅰ粗品，将所述宝藿苷Ⅰ粗品进行重结晶工艺处理，收集结晶体即得宝藿苷Ⅰ精制品。
  10. 根据权利要求9所述的宝藿苷Ⅰ的制备方法，其特征在于，当所述助溶剂为丙酮时，所述结晶工艺处理过程包括：将所述酶催化反应液于45-55℃水浴环境中减压蒸馏出丙酮，再将剩余溶液降温至10-15℃，待结晶体全部析出后过滤，滤饼经水洗、干燥处理后即得宝藿苷Ⅰ粗品。
  11. 根据权利要求9所述的宝藿苷Ⅰ的制备方法，其特征在于，所述重结晶工艺处理过程包括：将所述宝藿苷Ⅰ粗品溶解于无水乙醇中，并加入活性炭，搅拌并加热至55-65℃，趁热过滤，并用无水乙醇冲洗滤饼，滤液经减压蒸馏出乙醇后再加水，整个过程维持温度在55-65℃，之后降温至5-10℃，搅拌至结晶体全部析出后，过滤，滤饼经乙醇溶液冲洗、干燥处理后即得宝藿苷Ⅰ精制品。
  12. β-葡萄糖苷酶以及含有所述β-葡萄糖苷酶基因的微生物菌株在催化淫羊藿提取物、淫羊藿苷、朝藿定A、朝藿定B、箭藿苷A和箭藿苷B中的任意一种或多种制备宝藿苷Ⅰ中的应用，其特征在于：所述β-葡萄糖苷酶具有如SEQ ID NO：2所示的氨基酸序列，且所述β-葡萄糖苷酶特异性地将所述淫羊藿提取物中的淫羊藿苷、朝藿定A、朝藿定B、箭藿苷A和箭藿苷B全部水解成宝藿苷Ⅰ。
PCT/CN2018/084475 2018-04-25 2018-04-25 β-葡萄糖苷酶的用途及用其制备宝藿苷Ⅰ的方法 WO2019205026A1 (zh)

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